Vol. 10(36), pp. 3614-3624, 3 September, 2015 DOI: 10.5897/AJAR2015.10210 Article Number: EC3B4C055280 African Journal of Agricultural ISSN 1991-637X Copyright ©2015 Research Author(s) retain the copyright of this article http://www.academicjournals.org/AJAR
Full Length Research Paper
Growth performance and biochemical analysis of the genus Spirulina under different physical and chemical environmental factors
Dorothy Kemuma NYABUTO1,2, Kewei CAO1, Alfred Mugambi MARIGA3, Grace Wanjiru KIBUE1,2 , Meilin HE1 and Changhai WANG1*
1College of Resources and Environmental Science, Nanjing Agricultural University, No. 1 Weigang Road, Nanjing 210095, P. R. China. 2Department of Natural Resources, Egerton University, Egerton 536, Kenya. 3College of Food Science and Technology, Nanjing Agricultural University, No. 1 Weigang Road, Nanjing 210095, P. R. China.
Received 24 July, 2015; Accepted 21 August, 2015
Spirulina is useful to man in many aspects of life including health, food and cosmetics. In the present study, we aimed at investigating the optimum physical and chemical conditions that promote Spirulina mass production by varying a set of physical and chemical parameters, namely pH levels; Mg2+ ion concentration; nitrogen, phosphorous and carbon sources; salinity and different growing media. Temperature, light intensity, and light/dark cycle were maintained at 30°C±2, 4400 lx and 14:10 respectively throughout the study. At pH 9, the dry weight, protein, and chlorophyll a contents were 7.83±0.29 g/L, 1.34±0.12 mg/mL and 18.64±0.06 µg/mL respectively. At 0.8 mmol/L MgO, maximum dry weight, protein, and chlorophyll a contents were obtained. By using NaNO3 as the source of nitrogen, the dry weight was 2.24±0.13 g/L while the protein and chlorophyll a contents were 3.24±0.30 mg/mL and 2.53±0.24 µg/mL respectively. The highest biomass, chlorophyll a and protein contents were obtained by using K2HPO4 as the source of phosphorous. When NaHCO3 was used as the carbon source, the highest dry weight, protein and chlorophyll a yields were observed. Of the growing media used, Zarrouk’s medium yielded the highest biomass protein and chlorophyll a contents. Furthermore, under different salinities, the optimal dry weight, protein and chlorophyll a yields were obtained at 2.5%. This study provides the basis for high biomass production of Spirulina which is a promising microalga endowed with many health benefits due to its high protein content.
Key words: Biomass, chlorophyll a, protein, Spirulina.
INTRODUCTION
Spirulina platensis is a planktonic photosynthetic populations in tropical and subtropical water bodies that filamentous cyanobacterium that forms massive have high level of carbonate, bicarbonate and pH values
*Corresponding author. E-mail: [email protected] Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License Nyabuto et al. 3615
Table 1. Composition of Zarrouk’s medium (standard medium, SM).
Constituents Composition (g/L)
NaHCO3 18.0
NaNO3 2.5
K2HPO4 0.5
K2SO4 1.0 NaCl 1.0
CaCl2.2H2O 0.04
Na2EDTA 0.08
MgSO4.7H2O 0.2
FeSO4.7H2O 0.01 a A5 micronutrient sol. 1 mL/L
a A5 micronutrient solution consists of H3BO3, 2.86; MnCl2.4H2O, 1.81; ZnSO4.7H2O, 0.222; CuSO4.5H2O, 0.079; (NH4)6Mo7O24 (g/L).
Table 2. Composition of ES medium (West and McBride version).
Component Primary stock solution Quantity (1 L) Molar concentration in final medium -4 NaNO3 - 3.85 g 4.53×10 mol/L -5 Na2β-glycerophosphate.5H2O - 0.4 g 1.31×10 mol/L Fe-EDTA solution (see recipe below) 100 mL - Trace metal solution (see recipe below) 20 mL - -7 Thiamine (Vit. B1) 500 mg/L dH2O 8.0 mL 1.19×10 mol/L -8 Biotin (Vit. H) 50 mg/L dH2O 8.0 mL 1.78×10 mol/L -10 Cyanocobalomin (Vit. B12) 25 mg/L dH2O 3.5 mL 6.46×10 mol/L
Fe-EDTA solution consists of Na2EDTA.2H2O, 6.00 g/L; Fe (NH4)2(SO4)2.6H2O, 7.00 g/L. The trace metal solution consisted of Na2EDTA.2H2O, 2.548 g/L; H3BO3, 2.240 g/L; MnSO4.4H2O, 0.240 g/L; ZnSO4.4H2O, 0.044 g/L; CoSO4.7H2O, 0.010 g/L.
of up to 11. This cyanobacterium features morphological MATERIALS AND METHODS traits of the genus, that is, the arrangement of Microorganism multicellular cylindrical trichomes in an open lift-hand helix along entire length of the filaments (Soni et al., Cyanobacterium Spirulina used in the present study was purchased 2012). The genus Spirulina has gained importance and from Collection Centre of Marine Microalgae, Chinese Academy of international demand for its high phytonutrients Sciences, China. It was grown and maintained in 2 L sterilized value and pigments, which have applications in healthy Erlenmeyer flasks containing 1 L Zarrouk’s medium (Amala et al., foods, feeds, and therapeutics (Becker, 2007). It 2013), chemical composition of which is shown in Table 1, at room temperature, pH 9 with continuous illumination using cool white represents the second most important commercial fluorescent tubes whose light intensity was controlled and microalgae (after Chlorella) for the production of maintained at 2500 lx using the TES-1330A Digital Lux Meter (TES biomass used as healthy food and animal feed Electrical Electronic Corp, ISO 9001:2008). The experiment was (Sotiroudis et al., 2013). Its annual worldwide done in triplicate. Agitation was done thrice a day manually. All the production in the year 2000 was estimated to be reagents used were of analytical grade and were obtained from Shoude Experimental Equipment Co., Ltd, Nanjing. approximately 2000 tonnes (Spolaore et al., 2006). Spirulina has been used as food and nutritional supplements since antiquity (Falquet and Hurni, 2006). It Culture media and growing conditions is generally considered as a rich source of proteins, Zarrouk’s medium (Amala et al., 2013) was used as the standard vitamins, essential amino acids, minerals, essential fatty control medium (SM) and its constituents are shown in Table 1. It acids such as linolenic acid and sulfolipids was autoclaved (LDZX-75KBS Vertical Heating Pressure Steam (Mendes et al., 2003). In addition to poly-unsaturated fatty Sterilizer (Shanghai Shenan Medical Instrument Factory) at 121°C acids, it also has 6 poly unsaturated fatty acids, for 15 min and incubation was done aseptically. ES medium (West phycocyanin and other phytochemicals (Chamorro et al., and McBride Version) (West et al., 1999) was used as the growing medium in cultivation of Spirulina under different salinity levels and 2002). The present study reports the impact of different its constituents are shown in Table 2. The cyanobacterium Spirulina physical and chemical environment for mass production was grown under the following physical conditions: temperature 30 of Spirulina. ±2°C, light intensity 4400 lx and light/dark cycle 14:10. Agitation 3616 Afr. J. Agric. Res.
Table 3. Composition of CFTRI medium.
Ingredients g/L dH2O
NaHCO3 4.5
K2HPO4 0.5
NaNO3 1.5
K2SO4 1.0 NaCl 1.0
MgSO4.7H2O 1.2
CaCl2.2H2O 0.04
FeSO4.7H2O 0.01
Table 4. Composition of Bangladesh media No.3.
Ingredients g/L dH2O
NaHCO3 2.0 Urea 0.05 NaCl 1.0 Gypsum 1.5
Table 5. Composition of BG-11 (Blue Green-11) medium.
Stock solutions g/L dH2O
NaNO3 15.0
K2HPO4.3H2O 4.0
MgSO4.7H2O 7.5
CaCl2.2H2O 3.6 Citric acid 0.6 Ferric ammonium citrate 0.6 (Autoclave to dissolve) EDTA 0.1
Na2CO3 2.0
Trace metal mixture 1 ml, g/L
H3BO3 2.86
MnCl2.4H2O 1.81 Add each constituent ZnSO .7H O 0.222 separately to ~800 mL of dH2O 4 2 and fully dissolve between Na2MoO4.2H2O 0.39 each addition. Then make up CuSO4.5H2O 0.079 to 1 L Co(NO3)2.6H2O 0.0494
was done thrice a day and the experiments were done in triplicate. 3, 4, 5, 6, 7, and 1, respectively. All the sets of experiment were To achieve growth under different chemical conditions, the done in triplicate. Zarrouk’s medium was set at pH 7, 8, 9 (control), 10, 11 and 12; the MgO concentration was set at 0.0, 0.4, 0.8 (control), 1.2 and 1.6 mmol/L; for nitrogen source 2.5 g/L of NaNO3 (standard), urea and Cultivation NH4Cl were used; for phosphorus source 0.5 g/L of K2HPO4 (standard), KH2PO4 and NaH2PO4 were used and for the carbon Spirulina was cultivated aseptically in the 250 mL sterilized source 18 g/L of NaHCO3 (standard), Na2CO3 and K2CO3 were Erlenmeyer flasks containing 150 mL of respective sterilized media. used; salinities of 1.0, 1.5, 2.0, 2.5, 3.0, 3.5% were used and finally The temperature in the incubator was maintained at 30±2°C and CFTRI media, natural sea water, Bangladesh media No.3, BG-11, illuminated with day-light fluorescent tubes with light intensity at RaO’s, OFERR, and Zarrouk’s media (control) were used as the 4400 lx under Light/dark cycle, 14:10. During the process of growth, different growing media and their constituents are shown in Tables the flasks were shaken manually 3 times per day. The experiments Nyabuto et al. 3617
Table 6. Composition of RaO’s medium.
Ingredients g/L dH2O
NaHCO3 15.0
K2HPO4 0.5
NaNO3 2.5
K2SO4 0.6 NaCl 0.2
MgSO4.7H2O 0.04
CaCl2.2H2O 0.008 Fe-Ethylene diamine tetra acetate 0.2 Microelement solution 1 mL
The microelement solution consisted of H3BO3, 2.86 g/L; MnCl2.4H2O, 1.81 g/L; ZnSO4.7H2O, 0.222 g/L; CuSO4.5H2O, 0.079 g/L; (NH4)Mo7O24, 0.02 g/L.
Table 7. Composition of OFERR medium.
Ingredients g/L dH2O
NaHCO3 8.0 NaCl 5.0 Urea 0.2
K2SO4 0.5
MgSO4.7H2O 0.16
FeSO4.7H2O 0.05
H3PO4 0.052 mL
were run in triplicate. absorbencies of chlorophyll a containing supernatant were measured at 665 and 750 nm according to Oncel et al. (2006) and Soni et al. (2012). The algal biomass was harvested through Biomass yield filtration and then washed with distilled water and then frozen at - 20°C until use for chlorophyll a extraction according to Ritchie At 2-days intervals, 10 mL of homogenized algal suspension were (2006 and 2008),. Then later the frozen filter paper was crushed in filtered through a Whatman GB/T 1914 to 2007 filter paper that had a motor and pestle to homogenize the filter and algae. 5 to 10 mL of been pre-combusted in an oven for 1 h at a constant temperature of 100% ethanol was then added to the sample and centrifuged at 100°C and stored in a vacuum dessicator until use. The biomass 5000 r/min for 10 min. The absorbencies of chlorophyll a containing yield (the dry weight (g/l)) was determined by the method described supernatant were measured at 630, 647, 664, and 691 nm using by Moheimani et al. (2013) as described above. The biomass yield 100% ethanol as the blank. The protein content was determined by of the samples under different salinity levels was then calculated the Bicinchominic Acid (BCA) Protein Assay (Smith et al., 1985) through Optical density (OD) measurements using a using bovine serum albumin as the standard. 5 mL of homogenized spectrophotometer (SpectraMax M5 Microplate Reader, Molecular algal suspension was centrifuged at 3500 r/min for 5 min and later Devices, United States) and the growth rate (K′) was calculated mixed with 1Χ Phosphate Buffer Saline (PBS) (pH 7.4). The sample using the equation below (Van Leeuwe et al., 1997): was then sonicatend in an Ultra Sonic Crasher Noise Isolating Chamber (Ningbo Scientz Biotechnology Co. Ltd.). The K′ = ln (N2 / N1) / (t2 – t1) (1) absorbencies of protein containing supernatant were measured at 562 nm using bovine serum albumin as a blank. For estimation, the Where N1 and N2 are biomass at time 1 (t1) and time 2 (t2) following equations were used and expressed in mg/L per dry respectively. weight:
Chl a (mg/L) = 13.9 (A665–A750) (2) Biochemical analysis Chl a (µg/mL) = 0.0604 E630 – 4.5224 E647 + 13.2969 E664 – 1.7453 Chlorophyll a content was estimated according to Oncel et al. E691 (3) (2006), Soni et al. (2012) and Ritchie (2006 and 2008). The algal biomass was harvested through centrifugation and mixed with pure Protein content (µg/mL) = OD1/ OD2 × 563× dilution ratio (4) methanol, heated in 70°C water bath for 3 min for chlorophyll a extraction and later centrifuged at 3500 r/min for 5 min. The Where OD1 is the unknown sample, and OD2 is the standard 3618 Afr. J. Agric. Res.
Dry weight (g/L) 22 Chl a content (µg/mL) 20 Protein content (mg/mL) 18 16 14 12
Yield 10 8 6 4 2 0 7 8 9 10 11 12 pH levels
Figure 1. Cultivation of Spirulina under different pH levels
sample. in providing complete information related to growth, development and production of value added chemicals namely vitamins, amino acids, fatty acids, protein and Statistical analysis polysaccharides both in quantity and quality and The data obtained from the experiment were subjected to the disposing of carbon (II) oxide which is one of the major paired sample statistics (t-Test) using SPSS Software; version 14.0, causes of global warming (Bergman et al., 2013). 2007. The significance of the result was set at 5% level and Extensive research has been conducted on production of compared based on the mean values by least significant difference. Spirulina at salt lakes in the tropical regions (Sassano et al., 2004). Physico-chemical profile of Spirulina involves describing the relationship between growth and the RESULTS AND DISCUSSION environmental factors especially irradiance flux, density and temperature (Sotiroudis et al., 2013), which are Effect of different pH levels on biomass production of important in the evolution of micro algae and Spirulina cyanobacteria for biomass production, as well as their general characterization. High alkalinity is mandatory for pH is one of the main factors influencing the abundance the growth of Spirulina and bicarbonate is used to of inorganic carbon, dissolved total carbon (DTC). When maintain the high pH (Pandey et al., 2010; Jones et al., the pH is below 5, the majority of dissolved inorganic 1998). Sources of nutrition also affect the growth rate of - carbon (DIC) is CO2, which is equal to HCO3 at pH 6.6 cyanobacteria (Costa et al., 2001). The pH value of the - but at pH 8.3 almost all DIC is HCO3 (Ogbonda et al., culture medium combined with the dry cell weight may be 2007). Therefore, the pH should be controlled during an indirect method for determining the degree of cell cultivation to enhance the absorbability and utilization of growth of Spirulina. This is because the pH gradually CO2 by microalgae. The pH can also directly affect the rises as bicarbonate added to the culture medium is - permeability of the cell and the hydronium forms of the dissolved to produce CO2, which releases OH during the inorganic salt, and indirectly influence the absorption of cultivation of Spirulina (Moberg et al., 2012). The the inorganic salt. CO2 in the culture is consumed by the organism was adapted to six pH regimes (7, 8, 9, 10, 11 microalgae during photosynthesis, thereby increasing the and 12) in flask culture, monitored and yield expressed in pH of the medium. Therefore, substances like term of dry weight. The maximum bulk density of about hydrochloric acid and acetic acid have to be added to 7.83±0.29 g/L was observed when the pH of the culture control the pH to stop it from increasing beyond the medium was maintained at 9.0 with medium volume 150 tolerance of the microalgae. Compared with hydrochloric mL in a 250 mL flask as illustrated in Figure 1. The acid, acetic acid has the advantage of not only adjusting maximum bulk density was attained on the 25th day after the pH value but also acting as a carbon source to the inoculation of culture in Zarrouk’s medium. The enhance the growth rate of microalgae (Junying et al., increase in the yield of Spirulina could have been due to 2013). Culturing Spirulina in conical flask has its limitation the availability of mire space, oxygen, and light to the Nyabuto et al. 3619
Dry weight (g/L) 5.5 Chl a content (µg/mL) 5.0 Protein content (mg/mL) 4.5 4.0 3.5 3.0
2.5 Yield 2.0 1.5 1.0 0.5 0.0 0.0 0.4 0.8 1.2 1.6 MgO concentrations (mM)
Figure 2. Spirulina mass production under different Mg2+ ions concentrations.
culture flask and also due to the different solubility of CO2 ions toxicity on Spirulina, demonstrated a similar and other mineral compounds affected by the different pH phenomenon. levels. Earlier results also demonstrated that optimum pH for maximum growth of Spirulina was 9 to 9.5 ranges (Pandey et al., 2010). Spirulina is considered an Effect of different nitrogen, phosphorus, and carbon alkalophilic organism by nature (Jones et al., 1998). The sources to biomass production of Spirulina chlorophyll a content and protein content were maximum at pH 9 (Figure 1). Chlorophyll a content was 1.34±0.12 Many elements have to be provided for the growth of mg/mL while the protein content was 18.64±0.06 µg/mL. Spirulina, such as carbon (C), oxygen (O), hydrogen (H), Similar results have also been reported by various nitrogen (N), potassium (K), calcium (Ca), magnesium cyanobacteria researchers (Carvalho et al., 2002; (Mg), iron (Fe), sulfur (S), phosphorus (P) and trace Pandey et al., 2010). elements with the major nutrients being carbon, oxygen, hydrogen, nitrogen, phosphorus, and potassium. The first three are obtained from water and air while the latter Effect on different concentrations of Mg2+ ions to three have to be absorbed from the culture medium (Xin Spirulina biomass production et al., 2010). Nitrogen is one of the essential elements for the growth, development, reproduction, and other Magnesium oxide is known to hydrolyze in water to physiological activities of Spirulina. The nitrogen source generate hydroxide (Shand, 2006) and thus resulting to and concentration also affect the accumulation of lipid in increase in the pH level. In the present study, Spirulina. Usually, ammonium salts, nitrates, and urea concentrations of 0.0, 0.4, 0.8 (control), 1.2 and 1.6 are used as nitrogen sources, but their absorption rates mmol/L of nano-MgO were added into the growing and utilization are different (Xin et al., 2010). This is medium.0.8 mmol/L was set as the control measure since because ammonia is directly used to synthesize amino it is the amount in the Zarrouk’s media (Amala et al., acid while the other nitrogen sources have to be 2013). According to a study by Liu et al. (2012), nano- converted to ammonia to synthesize amino acid (Junying MgO is known to inhibit BOD and its toxicity results et al., 2013). It also has been found that microalgae grow primarily from the increase in pH level following MgO well with urea and nitrate. In the present study, sources hydrolysis thus the decrease in biomass yield in higher of nitrogen used included; NaNO3 (it is used as the concentrations. In terms of biomass yield, highest standard source of nitrogen), urea and NH4CL of the biomass was obtained at 0.8 mmol/L (control) (3.48±0.24 same osmolatity (0.03 mol/L). Highest biomass g/L) and lowest at 1.6 mmol/L (2.02±0.18 g/L) recordings were observed in NaNO3 nitrogen source respectively as illustrated in Figure 2. The low biomass (2.24±0.13 g/L) as illustrated in Table 8 while Chl a yield at the highest concentration could be attributed to protein contents were 2.53±0.24 and 3.24±0.30 mg/mL substrate toxicity. Wakte et al. (2011), studying metal respectively (Table 8). However, in urea and NH4CL, there 3620 Afr. J. Agric. Res.
Table 8. Spirulina cultivation under different nitrogen, phosphorus, and carbon sources.
Nutrient source Chl a content in Protein content in Nutrient Dry weight in g/L Final pH of culture (g/L) µg/mL mg/mL
2.5 NaNO3 (std) 2.24±0.13 10 2.53±0.24 3.24±0.30 Nitrogen 2.5 Urea N/D 10 N/D N/D
2.5 NH4Cl N/D 10 N/D N/D
0.5 K2HPO4(std) 2.08±0.17 10 2.52±0.21 2.93±0.06
Phosphorus 0.5 KH2PO4 1.85±0.16 10 1.44±0.13 2.30±0.07
0.5 NaH2PO4 0.80±0.07 10 1.30±0.07 2.28±0.22
18 NaHCO (std) 1.18±0.02 10 1.15±0.11 2.97±0.10 Carbon 3 18 Na2CO3 0.87±0.06 11 0.69±0.05 2.03±0.13
18 K2CO3 0.75±0.03 11 0.46±0.04 1.50±0.08
were no detections of biomass increase as a result of a represents 60% of the cost of nutrients (Alava et al., + high concentration of NH4 which is not suitable for 1997), which is one of the reasons for studying the growth as respiration of Spirulina is adversely affected by effects of different carbon source and their concentrations + too high concentration of NH4 ions (Chen et al., 2011). and finding alternative sources of this nutrient, such as Thus, NaNO3 is a better recommendation for nitrogen molasses (Andrade et al., 2008), residual CO2 (Ferreira source. et al., 2012) and synthetic CO2 (Rosa et al., 2011). The Phosphorus is another essential element for the concentration of dissolved inorganic carbon in anaerobic cultivation of Spirulina. Phosphate, hydrogen phosphate, effluent is lower than the amount indicated in the and other phosphates, play an important role in the formulation of standard culture media for production of metabolic processes of microalgae, as well as the Spirulina biomass, such as Zarrouk medium (Amala et succession of phytoplankton in aquatic ecosystems. al., 2013). In the current work, sources of C used were Phosphorus takes part in many metabolic processes, NaHCO3 (it is used as the standard source of nitrogen), such as signal transduction, energy conversion and Na2CO3 and K2CO3 of the same osmolatity (0.21 mol/L). photosynthesis (Navarro et al., 2008). The metabolic Maximum yield was observed with NaHCO3 carbon mechanisms of P in the different forms are different in source (1.18±0.92 g/L) as shown in Table 8. Chl a and microalgae. Orthophosphate is most easily absorbed and protein contents were 1.15±0.11 µg/mL and 2.97±0.10 significantly promotes the growth of microalgae (Navarro mg/mL respectively while the lowest biomass et al., 2008). According to the current work, sources of P concentration was observed when NaHCO3 was replaced 2- used were K2HPO4 (it is used as the standard source of by K2CO3 (0.75±0.52 g/L) due to less CO3 in the nitrogen), KH2PO4 and NaH2PO4 of the same osmolatity medium which are responsible for low alkalinity of the (0.003 mol/L). Optimal biomass yields were observed in growing medium (Table 8). K2HPO4 (2.08±0.17 g/L) as shown in Table 8. Chl a content (2.52±0.21 µg/mL) and protein content (2.93±0.06 mg/mL) are illustrated in Table 8. The lowest Effect of different salinities in biomass production of biomass concentration was observed when K2HPO4 was Spirulina replaced by NaH2PO4 (0.80±0.07 g/L) as illustrated in Table 8 and this is due to the changing of the N:P ratio, Salinity is the presence of high levels of soluble salts in as too high concentrations inhibited cell division of soils or waters. Management of salinity is important, as Spirulina thus the low biomass attained (Chu et al., elevated salt levels can have detrimental effects on 2013). Microalgae are phototrophic microorganisms, and production and the environment (Rafiqul et al., 2003). they include prokaryotic photosynthetic bacteria, called Spirulina can readily adapt to the environment, and it can cyanobacteria (Borges et al., 2013). These inhabit lake, creek, reservoir, or oceans, which have microorganisms have been investigated for their potential appropriate alkalinity, especially the southern ocean to enrich foods. The main nutrient required for Spirulina enjoying tropical climate. Additionally, seawater Spirulina cultivation is carbon, because the cells contain about performs better than freshwater Spirulina because of its 50% (w/w) of this element. Thus, the carbon source is the excellent nutrition content. most expensive component of Spirulina production. For In the present study artificial seawater medium, ES autotrophic growth (which is more suitable for large-scale medium (West and Mcbride Version) (West et al., 1999), open cultivation), carbon can be provided as CO2, was used as the growing medium and its constituents are carbonate or bicarbonate. If bicarbonate is used, it shown in Table 9. Its salinity levels were 1.0, 1.5, 2.0, 2.5 Nyabuto et al. 3621
Table 9. Biomass production of Spirulina under different salinities.
Sample number Salinity levels (%) Dry weight in g/L Chl a content in mg/L Protein content in µg/mL 1 1.0 0.88±0.07 1.34±0.12 131.57±3.00 2 1.5 0.97±0.03 1.41±0.12 129.33±2.00 3 2.0 1.23±0.10 1.50±0.14 127.40±1.00 4 2.5 (control) 1.44±0.06 1.86±0.13 117.77±2.00 5 3.0 0.77±0.08 1.31±0.12 109.11±4.50 6 3.5 0.69±0.08 1.00±0.20 85.68±3.50
Table 10. Specific growth rate (K′) of the cell.
S/N Salinity level (%) Specific growth rate of the cell (µm/day) 1 1.00 0.17 ±0.01 2 1.50 0.18 ±0.01 3 2.00 0.19 ±0.01 4 2.50 0.22 ±0.01 5 3.00 0.13 ±0.01 6 3.50 0.12 ±0.02
(control), 3.0 and 3.5% as shown in Table 9. To reduce contradictory to those reported earlier by some the cost of production of Spirulina, many methods have researchers. Most researchers have found similar results been attempted. The best method to date involved the (Shimamatsu, 2004; Zeng et al., 1998) whereas some use of nutrient-enriched seawater culture medium like the found an augmentation of protein contents with salinity one used in this current study (Rafiqul et al., 2003). In the increase (Rosales et al., 2005). However, strains and early stages of growth, there was an increase in biomass culture conditions were very different for all these studies to 1.44±0.06 g/L (Table 9) and the specific growth rate so the results are difficult to compare. In this work, it (K′) of Spirulina (0.22±0.01 µm/d (Table 10) respectively could be suggested that stressed cells had a lower in the 12th day at 2.5% salinity. As the salinity increased protein synthesis (at 3.5%; 85.68±3.50 µg/mL) capacity beyond 2.5%, biomass and growth rate decreased linked to the higher carbohydrate metabolism though the range of salinity was not high enough to inhibit (Kebede,1997; Rosales et al., 2005). As protein content growth. The decrease in growth with increasing salinity is a very important parameter for nutritional uses of has frequently been reported in the literature of Rosales Spirulina, the values have to be compared to the range of et al. (2005); Shimamatsu (2004), Zeng et al. (1998) and protein in Spirulina products found on the market, which (Kebede (1997). It is accompanied by a decrease in is 50 to 65%. The maximal value near 50%, obtained for photosynthetic efficiency, phycobilin/Chl a ratio and PSII 1.0% is satisfactory for food applications. activity and an increase in carbohydrate metabolism (Shimamatsu, 2004; Warr et al., 1985). In the present study, chlorophyll a content was stimulated at lower Biomass cultivation of Spirulina using different liquid salinities with the highest content being observed at 2.5% media (1.86±0.13 mg/L) followed by significant decrease at high salinity levels (Table 9). This could be as a result of Large-scale production of Cyanobacterial biomass is enhanced respiration that indicates that the response to essentially a complex process involving a large number salinity stress is an energy consuming process (Zeng and of variables and for their successful growth; the Vonshak, 1998), but the mechanisms involved have not environment needs to be conditioned to meet as many of yet been elucidated. It is probable that salinity stress the essential requirements of the organism. Among the affects light utilization and metabolism (particularly several constraints to the multiplication of cyanobacteria carbohydrates involved in osmoregulation) to counteract physical, physiological and economic limitations are of ionic and osmotic stresses (Kebede, 1997; Rosales et al., major importance. In tropical countries, especially 2005). Protein content was also influenced by the salinity developing countries such as India, emphasis is placed of the medium (Table 9). There was a drastic decrease in more on the production costs (Larsdotter, 2006; Raoof et protein content with increase in salinity levels. However, al., 2006). Culture of Spirulina in conical flask has its protein content results in this study are somewhat limitation in providing complete information related to 3622 Afr. J. Agric. Res.
6.0 Dry weight (g/L) 5.5 Chl a content (µg/mL) 5.0 Protein content (mg/mL) 4.5 4.0 3.5 3.0
Yield 2.5 2.0 1.5 1.0 0.5 0.0 Zarrouk's CFTRI Sea Water Bangladesh BG-11 RaO's OFERR Media No.3 Different liquid media (g/500mL)
Figure 3. Biomass production of Spirulina using different liquid media.
growth, development and production of value added 5.26±0.41 mg/mL on Zarrouk medium, 0.55±0.05 mg/mL chemicals namely vitamins, amino acids, fatty acids, on RaO’s medium, 3.10±0.23 mg/mL on CFTRI medium, protein and polysaccharides both in quantity and quality 4.23±0.42 mg/mL on OFERR medium, 1.33±0.01 mg/mL and disposing of carbon dioxide one of the major causes on Bangladesh medium No. 3, 2.26±0.11 mg/mL on of global warming (Bergman et al., 2013; Ferreira et al., natural sea water and 1.84±0.02 mg/mL on BG-11 2004). Extensive research has been conducted on medium respectively (Figure 3). Similar studies were production of Spirulina living at salt lakes in the tropical conducted by Hall et al. (2004), Bharat et al. (2011) and regions (Sassano et al., 2004). In addition, sources of Bharat et al. ( 2011). nutrition also affect the growth rate of Spirulina (Sotiroudis et al., 2013; Pandey et al., 2010). In the present study we investigated comparative growth rate of Conclusions Spirulina on CFTRI medium (Pandey et al., 2010), Natural seawater, Bangladesh Media No.3 (Bharat et al., Standardization of Spirulina in different media was 2011), BG-11 medium (Ruiz et al., 2004), RaO’s medium summarized and maximum growth noticed in Zarrouk’s (Devanathan et al., 2013), OFERR medium (Devanathan media. It is after the treatment of different pH that the et al., 2013) and Zarrouk’s medium (Amala et al., 2013) best growth resulted at pH 9. Mg2+ ion concentration as the control. The growth of Spirulina in flask culture effect was important for Spirulina cultivation as it is was then monitored and expressed in terms of dry known to hydrolyze in water to generate hydroxide and weight. Figure 3 shows that the specific dry weight of thus resulting to increase the pH level, which favors the Spirulina was 3.48±0.34 g/L on Zarrouk’s medium, growth of Spirulina. Biomass production of Spirulina was 1.80±0.17 g/L on RaO’s medium, 2.15±0.12 g/L on higher in media containing NaNO3 as the source of CFTRI medium, 2.30±0.18 g/L on OFERR medium, nitrogen, K2HPO4 as the source of phosphorus and 1.49±0.12 g/L on Bangladesh medium No. 3, 1.94±0.06 NaHCO3 as the source of carbon. Spirulina inoculated in g/L on natural sea water and 1.59±0.14 g/L on BG-11 2.5% salinity medium recorded the highest biomass yield. medium respectively. The data shows that specific The result of this investigation shows that both the growth of Spirulina is higher on Zarrouk’s medium. The physical and chemical environmental factors are very chlorophyll a content of Spirulina was 5.18±0.43 µg/mL important in biomass production and also chlorophyll a on Zarrouk’s medium, 0.04±0.00 µg/mL on RaO’s and protein biosynthesis in the Spirulina species. medium, 1.50±0.14 µg/mL on CFTRI medium, 3.39±0.27 µg/mL on OFERR medium, 0.13±0.01 µg/mL on Bangladesh medium No. 3, 0.56±0.05 µg/mL on natural Conflict of Interest sea water and 0.31±0.02 µg/mL on BG-11 medium respectively. The protein content of Spirulina was The authors have not declared any conflict of interest. Nyabuto et al. 3623
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